Wheat is the main staple food and largest grain crop of Pakistan. Abiotic stress reduces plant’s growth or development. An extended period of water deficits over months or years is the main cause of the drought. Drought arises in plants when the rate of transpiration is greater than rate of water uptake. QTLs (quantitative trait loci) are detected in mapping populations developed from controlled crosses segregating for the trait of interest and are a correlation of DNA marker data with phenotypic information. The research was conducted at PMAS Arid Agriculture University, Rawalpindi , Wheat Wide Crosses and Cytogenetic (WWC), National Agriculture Research Center, (NARC) Islamabad and Cereal Research institute (CRA-CER), Foggia Italy. The double haploid mapping population 5 (DR.MP. 5), with its individuals having pedigree of (Doy 1 // Ae. Squarrosa (458) / 5 / Opata), was planted under control (Field, fully irrigated) and stress (rain shelter) conditions. The parents of mapping population, i.e., SH349 (D genome grounded drought tolerant synthetic hexaploid wheat) and bread wheat (Opata M-85, drought susceptible) were also grown individually in the field and tunnel. The biochemical analysis included osmolytes determination, i.e. soluble sugar; proline and antioxidant included superoxide dismutase activity, cell membrane stability followed by molecular diagnostics including DNA extraction, polymerase chain reaction and capillary electrophoresis. Yield and Yield Components were included Days to heading, Days to physiological maturity, Plant height (cm), Spike length (cm), Number of grains per spike, 1000 Grain weight (g), pubescence, awn color and prostrate vs. erect habit. In this study, STATISTIX software was used for Two Factors Factorial Analysis of Variance, LSD (Least Significant Difference) did a2 comparison of means, and STATISTICA software was used to find out the correlation among different traits under control and stress conditions. For linkage and QTL analyses, JoinMap4 and MapQTL5 were used. Five Drought tolerant wheat lines were identified (18, 34, 54, 56 and 59). A linkage map was built by using SSR markers. One hundred and seventy four SSRs markers were used to screen the parents, (Opata, drought sensitive, and SH349, drought tolerant). Seventy-nine polymorphic markers were identified and applied to drought mapping populations. Interval mapping (IM) and Multiple QTL mapping (MQM) did detection of QTLs. In IM analysis, fifteen QTLs were found for stress condition and thirty-eight QTLs were found in control conditions. In MQM analysis, twelve QTLs were found to control under field conditions and sixteen QTLs were detected in MQM analysis under stress. Eleven Novel QTLs have been identified for different traits, six by IM and five by MQM. In the end the results of this research presented that there are some drought tolerant or resistant alleles have been identified which can be moved to vulnerable genotypes using MAS (Marker Assisted Selection). In addition, this study provided additional evidence that the QTL strategy is useful and able to enhance performance of existing cultivars. Further QTL studies will assist in the contribution of positive allelic diversity in the future.